Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation.

Tissue factor (TF) possesses additional physiological functions beyond initiating the coagulation cascade. Cellular signals initiated by cellular TF or on contact with TF‑containing microvesicles, contribute to wound healing through regulating a number of cellular properties and functions. TF regulates the cell cycle checkpoints, however the underlying signalling mechanisms have not been determined. Endothelial (human dermal blood endothelial cells and human umbilical vein endothelial cells) and epithelial [human telomerase reverse transcriptase‑human pancreatic nestin‑expressing ductal cells (hTERT‑HPNE) and AsPC‑1] cells were exposed to different concentrations of recombinant TF, and the influence on G1/S checkpoint regulators examined. Short‑term exposure to a lower concentration of TF promoted increased p16^INKa and decreased p21^CIP1/WAF1 expression, together with higher early region 2 binding factor (E2F) transcriptional activity and increased phosphorylation of Thr821/826 within retinoblastoma protein, leading to cell proliferation. The increase in p16^INKa expression was prevented following inhibition of β1‑integrin, or blocking the exosite within TF with AIIB2 and 10H10 antibodies, respectively. Exposure of cells to higher concentrations of TF induced disproportionate increases in p16^INKa and p21^CIP1/WAF1 expression, reduced retinoblastoma protein phosphorylation and E2F activity. Prolonged treatment of the immortalised hTERT‑HPNE cells with recombinant TF, resulted in significant downregulation of p16^INKa protein, which was partially due to reduced mRNA expression, together with increased E2F activity, and cyclin E mRNA expression. Although an increase in the methylation of the p16^INKa promoter was detected, the reduction in p16^INKa protein was concurrent with, and partly attributed to increased p14^ARF expression. TF appears early at the site of trauma, and its concentration is an ideal gauge for determining the extent of cellular damage, initiating clearance and repair. It is hypothesised that the balance of this signal is also dependent on the ability of cells to moderate the TF, and therefore on the level of damage. However, prolonged exposure of cells for example due to inflammation, leads to the dysregulation of the G1/S checkpoint by the tumour suppressors, leading to aberrant growth.